Immunity requires the random assembly of a diverse repertoire of immune receptors during lymphocyte development, many of which are self-reactive. To prevent autoimmunity, self-reactive lymphocytes are eliminated in central lymphoid organs during development. However, central tolerance mechanisms are not sufficient to veto all of the autoreactivity. In the first funding period of this program project award we developed a means to specifically target antigens to a subset of dendritic cells (DCs) that express the DEC- 205 antigen (CD8+DEC205+DCs) in vivo and showed that the steady state function of these cells is to maintain tolerance by inducing T cell deletion, or anergy or by facilitating the development of regulatory T cells. In preliminary experiments we have extended this antigen delivery method to a second group of DCs (CD8-33D1+DCs) that represent most of the DCs in mouse spleen. We find a novel form of regulation of antigen processing and presentation by DCs in vivo not anticipated by in vitro studies. CD8+DEC205+DCs are specialized for intracellular retention of immunologically intact antigen and presentation on MHCI, whereas CD8-33D1+DCs rapidly process the same antigen for presentation by MHCIL The long-range goals of this proposal are to determine how the two DC subsets contribute to maintaining self-tolerance and to exploit differences between the subsets in the prevention and therapy of autoimmunity. To accomplish these goals we propose to determine the cellular and molecular basis for the observed differences in antigen processing and presentation by the two subsets and to compare the two DC subsets in induction of T cell deletion, anergy and regulatory T cells. The working hypothesis is that the two DCsubsets will have unique and complementary functions in maintaining tolerance in vivo.